Sequential ChIP with a repeat of the sonication step? (View forum version)


Posted 27 July 2012 - 02:19 PM

Hello All,
First time poster here, so please forgive any faux pas. I am planning a rather complicated ChIP experiment where I would like to do a sequential ChIP with two different antibodies.

Here's the overall experimental objective (somewhat simplified and streamlined). I have a protein (Protein 1) that binds cellular DNA through it's C-terminal (most of the time) and through it's N-terminal (only when bound to a viral plasmid through it's C-terminal domain). I want to know what sequences it binds to with it's N-terminal domain while it is bound to the viral DNA. There is another protein (Protein 2) that binds to the viral plasmid about 1kb away from where Protein 1 binds. I want to first IP using an antibody against Protein 2 and then IP again using an antibody against Protein 1 and then sequence to see where Protein 1 binds through its N-terminus when it's C-terminus is attached to the viral plasmid. The catch, of course, is that there are a 1000bp of DNA between where Protein 1 and 2 bind on the viral plasmid.

I have outlined the steps as they are in my head below. I am wondering if this will work, or if I am missing something very obvious and it will be a fool's errand.
1. Crosslink cells
2. Sonicate to get fragments of about 3kb and IP with Antibody 1 (I use Protein G Dynabeads)
3. Elute the protein-DNA complex off of the beads
4. Sonicate to get fragments of about 200bp and IP with Antibody 2
5. Elute, reverse the crosslinks and prep for high-throughput sequencing.

Thank you in advance for any help you might be able to provide!


Posted 30 July 2012 - 02:20 PM

Depending on how you perform your first elution, you might encounter problems in the second shearing step. It might be worth performing the ChIP-ReChIP and then checking for enrichemnt of the viral sequence the protein is known to bind. Also the quality of the antibodies need to be excellent to maximize your first recovery and efficently grab everything in your second IP................I assume that the two proteins are viral? and thus made in-house? might have to check whether they work in a normal ChIP first...........there are quality commercial antibodies out there for certain viruses (i.e.-herpesviruses) just in case.